Interferon was discovered by Isaacs and Lindenmann, who noticed a factor interfering with influenza A virus when a chicken was infected with the virus, in 1957 (Isaacs, K. and Lindenmann, J., Proc. R. Soc. Lond., B147, 258-267 (1957)). Human interferons are proteins known as cytokines that allow communication between cells so that the protective defenses of the immune system that eradicate pathogens such as viruses can be triggered. Depending on the types of cells that release them, interferons are classified into interferon alpha, interferon beta and interferon gamma (Kirchner, H., et al., Tex. Rep. Biol. Med., 41, 89-93 (1981): Stanton, G. J., et al., Tex. Rep. Biol. Med., 41, 84-88 (1981)). That is, interferon alpha is released from B lymphocytes, interferon beta from null lymphocytes and macrophages, and interferon gamma from T lymphocytes with the aid of macrophages. Interferons were reported to exhibit anti-viral activity, anti-cancer activity, the activation of NK (natural killer) cells, and synergistic inhibitory action on mycelocyte growth (Klimpel, et al., J. Immunol., 129, 76-78 (1982); Fleischmann, W. R., et al., J. Natl. Cancer Inst., 65, 863-966 (1980); Weigent., et al., Infec. Immun., 40, 35-38 (1980)). Since then, a large number of studies have discovered that in addition to exhibiting anti-viral effects, interferons act as regulatory factors in the expression, structure and functions of genes within cells, especially direct anti-proliferative effects. Further, a function of interferons is to fight various diseases caused by infections and various tumors.
Interferon alpha is produced in leukocyte cells after exposure to mitogen, viruses or tumor cells. To date, a multigene family of at least 20 genes have been discovered for interferon alpha and are known to encode polypeptides most of which consist of 165 or 166 amino acids.
Clinical tests have demonstrated that recombinant human interferon alpha is effective at treating various solid cancers. Particularly, interferon alpha is known to have effective therapeutic effects on bladder cancer, kidney cancer, and AIDS-associated kaposi's sarcoma (Torti, F. M., J. Clin. Oncol., 6, 476-483 (1988); Vugrin, D., et al., Cancer Treat. Rep., 69, 817-820 (1985); Rios, A., et al., J. Clin. Oncol., 3, 506-512 (1985)). Further, a recent report mentions that interferon alpha is therapeutically applicable to the treatment of hepatitis type C (Davis, G. G., et al., N. Engl. J. Med., 321, 1501-1506 (1989)). Based on these new findings, the therapeutic area of interferon alpha has become wider.
Polypeptides such as interferon alpha tend to easily denature due to their low stability, be degraded by proteolytic enzymes in the blood and to be easily passed through the kidney or liver. Thus, protein drugs, including polypeptides as pharmaceutically effective components, need to be frequently administered to patients to maintain the desired blood level concentrations and titers. However, such frequent administration of protein drugs, most of which are in injection form, causes pain to patients.
To solve these problems, a lot of effort has been put into improving the serum stability of protein drugs and maintaining the drugs in the blood at high levels for a prolonged period of time to maximize the pharmaceutical efficacy of the drugs. For use as long-acting preparations, protein drugs must be formulated to have high stability and have their titers maintained at sufficiently high levels without incurring immune responses in patients.
A conventional approach to stabilizing proteins and preventing enzymatic degradation and clearance by the kidneys is to chemically modify the surface of a protein drug with a polymer having high solubility, such as polyethylene glycol (PEG). By binding to specific or various regions of a target protein, PEG stabilizes the protein and prevents hydrolysis, without causing serious side effects (Sada et al., J. Fermentation Bioengineering 71: 137-139). However, despite its capability to enhance protein stability, PEGylation has problems such as greatly reducing the titers of physiologically active proteins. Further, there is a decrease in the yield with increasing molecular weight of the PEG due to the reduced reactivity of the proteins.
Another alternative strategy for improving the in vivo stability of physiologically active proteins is to link a gene of a physiologically active protein to a gene encoding a protein having high serum stability with the aid of genetic recombination technology and culturing the cells transfected with the recombinant gene to produce a fusion protein. For example, a fusion protein can be prepared by conjugating albumin, a protein known to be the most effective in enhancing protein stability, or its fragment to a physiologically active protein of interest by genetic recombination (PCT Publication Nos. WO 93/15199 and WO 93/15200, European Pat. Publication No. 413,622).
Another method is to use an immunoglobulin as described in U.S. Pat. No. 5,045,312 wherein human growth hormone is conjugated to bovine serum albumin or mouse immunoglobulin by use of a cross-linking agent. The conjugates have enhanced activity, compared with unmodified growth hormone. Carbodiimide or glutaraldehyde is employed as the cross-linking agent. Non-specifically bonding to the peptides, however, such low-molecular weight cross-linking agents do not promise the formation of homogeneous conjugates and are even toxic in vivo. Further, the activity enhancement that the patent is responsible for takes place only because of chemical coupling with the growth hormone. The method of the patent cannot guarantee enhanced activity for various kinds of polypeptide drugs, so that the patent does not recognize even protein stability-related factors, such as duration, the blood half-period, etc.
As such, a long-acting protein drug formulation with improved in vivo duration and stability is required. For use in the long-acting drug formulation, protein conjugates in which a physiologically active polypeptide is covalently linked to a non-polypeptide polymer and an immonoglobulin Fc region have recently been suggested in Korean Patent Nos. 10-0567902 (Physiologically active polypeptide conjugate having improved in vivo durability) and 10-0725315 (Protein complex using an immunoglobulin fragment and method for the preparation thereof).
To apply long-acting interferon alpha conjugates to drug products, it is necessary to maintain the pharmaceutical efficacy thereof in vivo while restraining physicochemical changes such as light-, heat- or additive-induced degeneration, aggregation, adsorption or hydrolysis during storage and transportation. Long-acting interferon alpha conjugates are more difficult to stabilize than interferon alpha polypeptide itself because they are increased in volume and molecular weight.
On the whole, proteins have a very short half life and, when exposed to unsuitable temperatures, water-air interfaces, high pressures, physical/mechanical stress, organic solvents, microbial contamination, etc., they undergo degeneration in the forms of the aggregation of monomers, precipitation by aggregation, and adsorption onto the surface of containers. When degenerated, proteins lose their inherent physicochemical properties and physiological activity. Once degenerated, proteins almost cannot recover their original properties because the degeneration is irreversible. Particularly in the case of proteins administered in a dose of as small as hundreds of micrograms per injection, such as interferon alpha, when they lose stability and thus are absorbed onto the surface of the container, a relatively great amount of damage results. In addition, absorbed proteins easily aggregate during a degeneration process, and aggregates of the degenerated proteins, when administered into the body, act as antigens, unlike proteins synthesized in vivo. Thus, proteins must be administered in a sufficiently stable form.
Many methods have been studied to prevent the degeneration of proteins in solutions (John Geigert, J. Parenteral Sci. Tech., 43(5): 220-224, 1989; David Wong, Pharm. Tech., October, 34-48, 1997; Wei Wang., Int. J. Pharm., 185: 129-188, 1999; Willem Norde, Adv. Colloid Interface Sci., 25: 267-340, 1986; Michelle et. al., Int. J. Pharm. 120: 179-188, 1995).
To achieve the goal of stability, some protein drugs are subjected to lyophilization. However, lyophilized products are inconvenient because they must be re-dissolved in injection water to be used. In addition, they require that a massive investment be made in large-capacity freeze-driers because lyophilization is included in the production process thereof. The contraction of proteins with the use of a spray drier has also been suggested. However, this method is economically unfavorable due to low production yield. Further, a spray-drying process exposes the proteins to high temperature, thus having a negative influence on the stability of the proteins.
As an alternative to overcoming the limitations, stabilizers have appeared that, when added to proteins in solution, can restrain physicochemical changes of protein drugs and maintain in vivo pharmaceutical efficiency even after having been stored for a long period of time. Among these are carbohydrates, amino acids, proteins, surfactants, polymers and salts. Inter alia, human serum albumin has been widely used to stabilize various protein drugs, and its performance in this respect has been verified (Edward Tarelli et al., Biologicals, 26: 331-346).
A typical purification process of human serum albumin includes inactivating biological contaminants such as mycoplasma, prion, bacteria and virus or screening for or examining for the presence of one or more biological contaminants or pathogens. However, there is always the risk of exposing patients to the biological contaminants because they were not completely removed or inactivated. For example, human blood from donors is screened to examine whether it contains certain viruses. However, this process is not always reliable. Particularly, certain viruses existing in a very small number cannot be detected.
Further, different proteins may be gradually inactivated due to the chemical differences thereof because they are subjected to different ratios and conditions during storage. The effect of a stabilizer on the storage term of proteins differs from one protein to another. That is, various stabilizers may be used at different ratios depending on physicochemical properties of the proteins of interest. When concurrently used, different stabilizers may bring about reverse effects due to competition and the erroneous operation thereof. A combination of different stabilizers also elicits different effects because they cause the proteins to change in characteristics or concentration during storage. Because the suitability of the stabilizing activity of each stabilizer is relative to a given range of concentration, care must be exercised when combining different kinds and concentrations of different stabilizers.
Particularly, as pertains to long-acting interferon alpha conjugates which have improved in vivo duration and stability, the molecular weights and volumes thereof are quite different from those of general interferon alpha because they are composed of the physiologically active peptide interferon alpha, non-peptide polymers, and the immunoglobulin fragment Fc. Moreover, the physiologically active peptide interferon alpha and the immunoglobulin fragment Fc must be stabilized simultaneously because both of them are peptides or proteins.
As stated above, different proteins may be gradually inactivated due to the chemical differences thereof because they are subjected to different ratios and conditions during storage. In addition, different stabilizers suitable for respective peptides or proteins, when concurrently used, may incur adverse effects rather than the desired effects, due to competition and the erroneous operation thereof.
Accordingly, it is difficult to establish stabilizer compositions for long-acting interferon alpha conjugates, which are designed to simultaneously stabilize both interferon alpha and an immunoglobulin Fc region.